Related Experiment Video
Updated: Apr 15, 2026
Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets
Published on: November 29, 2024
Super-resolved calibration-free flow cytometric characterization of platelets and cell-derived microparticles in
Anastasiya I Konokhova1, Darya N Chernova1,2, Alexander E Moskalensky1,2
1Institute of Chemical Kinetics and Combustion SB RAS, 630090, Novosibirsk, Russia.
Abstract:
Importance of microparticles (MPs), also regarded as extracellular vesicles, in many physiological processes and clinical conditions motivates one to use the most informative and precise methods for their characterization. Methods based on individual particle analysis provide statistically reliable distributions of MP population over characteristics. Although flow cytometry is one of the most powerful technologies of this type, the standard forward-versus-side-scattering plots of MPs and platelets (PLTs) overlap considerably because of similarity of their morphological characteristics. Moreover, ordinary flow cytometry is not capable of measurement of size and refractive index (RI) of MPs. In this study, we 1) employed the potential of the scanning flow cytometer (SFC) for identification and characterization of MPs from light scattering; 2) suggested the reference method to characterize MP morphology (size and RI) with high precision; and 3) determined the lowest size of a MP that can be characterized from light scattering with the SFC. We equipped the SFC with 405 and 488 nm lasers to measure the light-scattering profiles and side scattering from MPs, respectively. The developed two-stage method allowed accurate separation of PLTs and MPs in platelet-rich plasma. We used two optical models for MPs, a sphere and a bisphere, in the solution of the inverse light-scattering problem. This solution provides unprecedented precision in determination of size and RI of individual spherical MPs-median uncertainties (standard deviations) were 6 nm and 0.003, respectively. The developed method provides instrument-independent quantitative information on MPs, which can be used in studies of various factors affecting MP population.
Insights
Scanning flow cytometry (SFC) offers precise characterization of microparticles (MPs) and extracellular vesicles. This new method accurately separates MPs from platelets and determines their size and refractive index with high accuracy.
Area of Science:
- Biotechnology
- Analytical Chemistry
- Cell Biology
Background:
- Microparticles (MPs), also known as extracellular vesicles, play crucial roles in physiological and clinical processes.
- Accurate characterization of MPs is essential for understanding their functions.
- Standard flow cytometry methods struggle to differentiate MPs from platelets due to overlapping light-scattering properties.
Purpose of the Study:
- To utilize scanning flow cytometry (SFC) for enhanced identification and characterization of MPs.
- To establish a reference method for high-precision measurement of MP morphology, including size and refractive index (RI).
- To determine the minimum MP size detectable by SFC using light scattering.
Main Methods:
- Equipped SFC with 405 nm and 488 nm lasers to capture light-scattering profiles and side scattering.
- Developed a two-stage method for accurate separation of platelets (PLTs) and MPs in platelet-rich plasma.
- Applied spherical and bispherical optical models to solve the inverse light-scattering problem for MP characterization.
Main Results:
- Achieved accurate separation of PLTs and MPs.
- Demonstrated unprecedented precision in determining individual spherical MP size (median uncertainty of 6 nm) and RI (median uncertainty of 0.003).
- Established the capability of SFC to characterize MPs down to a specific size limit via light scattering.
Conclusions:
- SFC provides a powerful tool for precise MP characterization.
- The developed method offers instrument-independent, quantitative data on MP size and RI.
- This advancement facilitates deeper research into factors influencing MP populations in various biological contexts.
